Serodiagnosis of Scrub Typhus with Antigens Immobilized on Nitrocellulose Sheet

This study was designed to develop a simple method for serodiagnosis of scrub typhus. The basis of the method is detection of anti-Rickettsia tsutsugamushi antibody in patient serum by reaction with antigens dot blotted on a nitrocellulose sheet (NCS). The final evaluation of the reaction is performed by observing the color intensity which develops as a result of sequential treatments of the NCS with peroxidase-conjugated anti-human immunoglobulin G or immunoglobulin M antibody and with the substrate of the enzyme. After various trials, we found that the best results were obtained by using a purified antigen which adhered to an NCS at 0.2 to 2 micrograms of protein per dot and a test serum diluted 1,000- to 4,000-fold. Under these conditions, almost all antibody-positive sera showed a distinct color at the dot on the NCS, so that a positive reaction could be distinguished by the naked eye from a negative reaction with antibody-negative sera, which developed only a faint color. A comparison of the results of screening of antibody-positive and -negative sera by this method and the immunofluorescence test showed that both methods produced similar results. From these results, it is concluded that this dot immunoassay can be useful for the serodiagnosis of scrub typhus.